Identification and Characterization of In-planta Expressed Secreted Effector Proteins from Magnaporthe oryzae Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Pattavipha Songkumarn, M.S. Graduate Program in Plant Pathology The Ohio State University 2013 Dissertation Committee: Dr. Guo-Liang Wang, Advisor Dr. Pierluigi (Enrico) Bonello Dr. Thomas K. Mitchell Dr. David M. Mackey Copyright by Pattavipha Songkumarn 2013 Abstract Rice blast disease, caused by the fungus Magnaporthe oryzae, is one of the most serious diseases of rice. Interactions between rice and M. oryzae involve the recognition of cellular components and the exchange of complex molecular signals from both partners. How these interactions occur in rice is still elusive. Previously, we employed gene expression profiling, including robust-long serial analysis of gene expression (RL-SAGE), massively parallel signature sequencing (MPSS), and sequencing by synthesis (SBS), to examine transcriptome profiles of infected rice leaves. A total of 6,413 in-planta expressed fungal genes, including 851 genes encoding putative effector proteins, were identified. To elucidate the molecular basis of interactions between rice and M. oryzae upon infection, we combined the results from gene expression profiling with high throughput gene cloning, and rapid protoplast transient expression assay for large-scale identification of M. oryzae effector proteins that induce cell death in rice plants. In total, seven M. oryzae proteins were found to induce cell death in rice protoplasts and six of them were found to induce cell death in the non-host plant, Nicotiana benthamiana. However, this cell death only occurred when the proteins contained the signal peptide for secretion to the extracellular space. Although these seven M. oryzae effectors are diverse in their sequence and structure, the physiological basis of cell death induced by most of these effectors is similar. Among all identified effectors, three effectors, i.e., MGG_05232, MGG_08370, MGG_08409, contain carbohydrate-binding modules (CBMs). To elucidate the ii pathogenesis function of the three CBM-containing effectors, we generated transgenic M. oryzae strains that carried out gene replacement and gene overexpression. Among the three of them, the MGG_08409 effector containing cellulose binding domain showed pathogenic function in rice. In addition, MGG_08409 binds to cellulose, the component of plant cell wall, and has glucanase activity against cellulose substrate. This study demonstrates that our integrative genomic approach is effective for the identification of in-planta expressed cell death- inducing effector from M. oryzae, which may play important role facilitating colonization and fungal growth during infection. Characterization of these fungal effector proteins will provide new insights into the molecular basis of the rice and M. oryzae interactions. iii Dedicated to my beloved family: Narong, Wanchern, Natinee and Peerapat Songkumarn iv Acknowledgements I would like to express my gratitude to my advisor, Dr. Guo-Liang Wang for giving me the opportunity, intellectual and providing supports. Your encouragement, guidance, understanding and patience have provided a good basis for the present thesis. In addition, I wish to thank my SAC members: Dr. Pierluigi (Enrico) Bonello, Dr. David M. Mackey, and Dr. Thomas K. Mitchell for their suggestions and assistance for my research. I also thank all Wang lab members who always be there whenever I need help. Thank you for your help, encouragement, and discussion during the development of my experiments. Thank for Dr. Songbiao Chen, I am very grateful and fortunate to have you as my mentor. My sincere thank goes to Maria, without invaluable friendly assistance, I could not complete my work. I am thankful to Gautam and Chanho, both of you are such great friends. Thank you very much for your continuous advice, encouragement and your precious friendship throughout my graduate program. I also thanks The Royal Thai Government and Kasetsart University for providing me all support as a Thai scholar. Finally, I would like to give special thanks to my family for their love and supports. They always stand by me. This thesis is simply impossible without them. v VITA 1993-1997……………………………………………….B.S. (Microbiology), Prince of Songkla University, Songkla, Thailand 1997-2001……………………….……………………….M.S. (Microbiology), Kasetsart University, Bangkok, Thailand 2001-2006……………………………………………….Lecturer, Nakhon Si Thammarat Rajabhat University, Nakhon Si Thammarat, Thailand 2006-2011………………………………………………Thai Government Scholar PUBLICATIONS Chen, S., Songkumarn, P., Venu, R., Gowda, M., Bellizzi, M., Hu, J., Liu, W., Ebbole, D., Mitchell, T., and Wang, G. L. (2012). Identification and Characterization of In-planta Expressed Secreted Effectors from Magnaporthe oryzae that Induce Cell Death in Rice. Mol. Plant- Microbe Interact. Inpress. (Co-first author) vi Park, C. H., Chen, S., Shirsekar, G., Zhou, B., Khang, C. H., Songkumarn, P., Ning, Y., Wang, R., Bellizzi, M., Valent, B., and Wang, G. L. (2012). The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ligase APIP6 for suppression of PAMP-triggered immunity in rice. Plant Cell 24, 4748-4762. Chen, S., Songkumarn, P., Liu, J., and Wang, G. L. (2009). A versatile zero background T-vector system for gene cloning and functional genomics. Plant Physiol. 150, 1111-1121. Chen, S. M. Gowda, R. C. Venu, P. Songkumarn, C. H. Park, M. Bellizzi, D. Ebbole, G. L. Wang. (2009). Isolation and functional analysis of putative effectors from M. oryzae using integrated genomic approaches. In Advances in Genetics, Genomics and Control of Rice Blast Disease, Eds. G.L. Wang and B. Valent, Springer, pp 93-103. FIELDS OF STUDY Major Field: Plant Pathology vii TABLES OF CONTENTS Abstract………………………………………………………………………………………………...……………………………………ii Dedication………………………………………………………………………………………………………………………………….iv Acknowledgements……………………………………………………………………………………………………………….……v Vita……………………………………………………………………………………………………………………………….……………vi List of figures…………………………………………………………………………………………………………………..…………ix List of tables…………………………………………………………………………………………………………………..…….….xiii Chapter 1 Introduction……………………………………………………………………………………………….………………1 Chapter 2 Identification and Characterization of In-planta Expressed Secreted Effectors from M. oryzae that Induce Cell Death in Rice…………………………………………………………………………..29 Chapter 3 Functional analysis of M. oryzae carbohydrate binding module (CBM)- containing effectors………………………………………………………………………………….………………………………70 Chapter 4 Conclusion and Future Challenges………………………………………………………….………………151 Bibliography……………………………………………………………………………………………………………………………157 Appendix.....................................................................................................................................191 viii LIST OF FIGURES 1.1 M. oryzae infection cycle and rice blast symptoms…………………………………………..…………………..5 1.2 Diagram of development of biotrophic interfacial complex (BIC) during rice-leaf sheath invasion………………………………………..………………………………………………………………………….……………….13 1.3 Summary statistics for gene expression profiling by RL-SAGE, MPSS and SBS, and identifying of in-planta expressed M. oryzae genes encoding putative secreted protein…………………….……..24 1.4 Gene expression profiling of M. oryzae during its interaction with rice……………………..………..26 1.5 Gene Ontology (GO) annotation of In-planta expressed M. oryzae putative secreted protein………………………………………………………………………………………………………………………………….….27 2.1. The improved rice protoplast isolation method…………………………….…………………………………..33 2.2. Construction of the ZeBaTA system………………………….…………………………………………….………….36 2.3. RT-PCR validation of putative effector genes……………………………………………………………………..50 2.4. Identification of five in-planta expressed putative secreted proteins that induce cell death in rice cells…………………………………………………………………………………………….……..…………………………..55 2.5. Transient expression of M. oryzae cell death inducing proteins in the non-host plant, N. benthamiana………………………………………………………………………………………………..………………………57 ix 2.6. In-planta expression pattern of the five MoCDIPs……………………………………………………..……….59 2.7. Validation of the secretory feature of the five MoCDIPs by yeast secretion analysis….………61 3.1. Schematic diagram of the major structures of a plant cell wall…………………….…………………….76 3.2. A diagram illustrating PCR amplifications of the components for MGG_05232 gene deletion by split marker………………………………………………………………………………………………………………..………..82 3.3. Schematic views of 5 MoCDIPs show structural analyses of MGG_03356, MGG_05531, MGG_07986, MGG_08409, and MGG_10234…………………………………………………….……………………..95 3.4. Schematic diagram of the strategy used for identification of M. oryzae genes encoding CBM- containing effector proteins………………………………..……………………………………………………………………97 3.5. Identification of M. oryzae CBM-containing proteins that induce cell death in rice cells…101 3.6. Transient expression of M. oryzae genes encoding for CBM-containing proteins in non-host plant, N. benthamiana……………………………………………..……………………………………………………………..104 3.7. In-planta expression pattern of the M. oryzae genes encoding CBM-containing proteins, MGG_05232 and MGG_08370………………….………………………………………………………...………………….107 3.8. Validation of the secretory feature of M. oryzae CBM-containing proteins, MGG_05232 and MGG_08370 by yeast secretion analysis………………………………………………………………….……………..109 3.9. Schematic views of MGG_05232 and